HU196464B - New process for producing 1,2-dehydrosteroids - Google Patents

Abstract

Through the use of microbial cells, air-dried with or without heating, that are able to catalyse I- dehydrogenation of steroids, there is a more efficient conversion of 1.2-saturated steroids to the corresponding 1.2 dihydro derivatives, which can be obtained with the best processes of the previous literature in an aqueous medium, particularly in the presence of an exogenous electron acceptor. A process is also described and claimed to add an aromatic hydrocarbon as a solvent that cannot be mixed with water to a cell suspension of A. simplex, an exogenous electron acceptor, and the steroid to be dehydrogenated. The suspension is agitated and oxygen is supplied so that the oxygen contents in the gaseous space is lower than the minimum concentration for combustion; or the reaction is performed below the inflammation point of the organic solvent.

Description

The present invention relates to a microbiological process for the preparation of 1,2-dehydro-β-steroids from their corresponding 1,2-saturated derivatives.

Corticosteroids were first used in the fifties for medicinal purposes, when cortisone acetate was used to treat rheumatoid arthritis. Subsequent studies have shown that the 1,2-unsaturated hydrocortisone and cortisone derivatives prednisolone and prednisone have a significant excess effect and cause less salt retention. Subsequently, most steroids used to treat corticoid-related diseases were prepared by having double bonds at the 1- and 2-positions.

In 1977, two US patents proposed a process based on sterol precursors for the synthesis of corticosteroids, which represented a new approach to the synthesis of corticosteroids. U.S. Pat. No. 4,035,236 relates to the preparation of 9alpha-hydroxy-androstenedione by fermentation of sitosterol, stigmasterol or cholesterol. U.S. Patent 4,041,055 relates to the general synthesis of pharmaceutically acceptable corticosteroids from this androstene derivative. The intermediates described in the process ^{3-keto-4-9 <u>} may be configuration.

Further references in the literature on the conversion of 1,2-saturated steroids to 1,2-dehydro-steroids include:

U.S. Patent No. 2,837,464 describes the preparation of diene with Corynebacterium. According to the description, 1-dehydrogenation of steroids is performed by fermentation using Arthrobacter (Corynebacterium) simplex.

U.S. Patent No. 3,360,439 relates to the preparation of 1-dehydro steroids. 1-Dehydrogenation of steroids is accomplished by pretreating A. simplex cells with lower alkanol or alkanone, such as acetone, and then mixing the substrate with a hydrogen transfer catalyst.

Charney, W., and Herzog, H., in Microbial Transformation of Steroids (Academic Press, Inc., New York, pp. 4-9 and 236-261 (1967)) provide a historical overview of steroid bioconversions and list Microorganisms known and used for 1-dehydrogenation.

Yamane, T., Nakatani, H., et al. (Biotechnology and Bioengineering, 21, 2133 (1979)) investigated 1-dehydrogenation of 4-androstene-3,17-dione with Nocardia rhodocrous in 50% benzene-heptane. The addition of this solvent to the bioconversion mixture increased the reaction rate 180-fold. However, benzene has a carcinogenic property. The preferred ecetron acceptor for Nocardia rhodocrous activity was phenazine methosulfate. This electron acceptor is too expensive and unstable to be used in bioconversion processes. Nocardia rhodocrous was inactive in the presence of menadione (2-methyl-1,4-naphthoquinone), an electron acceptor favorable for Arthrobacter simplex. The enzyme in Nocardia rhodocrous is different from the enzyme produced by A. simplex. The reaction was carried out in an organic solvent containing 20% steroid. It has been shown that with increasing concentrations of steroids, the reaction rate is reduced. Oxygen is an essential element of the 1-dehydrogenation reaction. - The solvent used (50% benzene-heptane) is flammable in the presence of oxygen. How oxygen was introduced into the system to prevent inflammation has not been reported.

Somoto, K., Jin, I. et al., Agricultural Biological Chemistry, 44, 1119-1126 (1980), investigated the 1-dehydrogenation of hydrocortisone with A. simplex. Two immiscible solvents, n-butanol and n-butanol. Amyl alcohol was used, but bioconversion did not occur in the presence of these solvents, and water soluble solvents such as 10% methanol and 10% propylene glycol were found to be the most suitable solvents.

In summary, the prior art references do not disclose or suggest improved methods of the present invention.

The present invention utilizes heat-dried microorganism cells capable of catalyzing the 1-dehydrogenation of steroids to convert 1,2-saturated steroids to the corresponding 1,2-dehydro derivatives by significantly better conversion than the exogenous electron in the aqueous medium described so far. reaction in the presence of an acceptor, where the yield was best, the reaction rate was the fastest.

Advantages of the process of the present invention over known microbiological steroid dehydrogenation processes:

(a) less unconverted steroids in the final product of bioconversion,

(b) the reaction may be carried out at higher steroid concentrations,

c) specific microorganism cells, such as A. simplex, are required for the conversion of the steroid,

(d) the process is less sensitive to contamination by steroid ban, in cells of the micro-organism or by cells in the steroid,

e) reduction or complete elimination of steroid degrading enzymes present in the microorganism cells can be achieved in higher yields.

Any known 1-dehydrogenating microorganism can be used in the process of the invention.

The l-dehydrogenating bacteria can be divided into two groups according to Bergey, Manual of Determinative Biology (8th edition). The method described herein can be successfully applied to Arthrobacter and Corynebacterium species and strains discussed in Part 17, "Actinomycetes and Related Microorganisms". 1-Dehydrogenating species of other strains, such as Nocardia, Mycobacterium, Streptomyces and Bacterium are also suitable for carrying out the process.

Our method is presented on two publicly available steroids on l-dehydrogenating microorganisms. On the one hand, Bacterium cyclooxydans (ATCC No. 12673), a 1-dehydrogenator, was examined in U.S. Patent 3,065,146. The microorganism Arthrobacter simplex (ATCC No. 6946) in U.S. Patent No. 2,837,464 is more commonly used for the 1-dehydrogenation of steroids. Therefore, for the most part, this microorganism is used to describe the process of the invention. However, it is to be noted that any 1-dehydrogenating microorganism is capable of carrying out the process of the invention.

The microorganisms were cultured on the following medium:

(a) inorganic compounds (nitrates or ammonium salts) or compounds containing organic nitrogen (yeast extract, peptone, cereal broth) to provide the nitrogen needed for growth,

(b) sources of carbon and energy, such as carbohydrates and sugars, oils, fatty acids and their methyl esters, alcohols, amino acids or organic acids;

(c) ions and trace elements (sodium, potassium, magnesium, phosphate, sulphate, manganese, copper, cobalt, molybdenum) in quantities present in tap water or in materials which are less refined.

Microorganisms require oxygen in the air above them to grow. Suitable temperatures for growth of A. simplex are 10-45 ° C, preferably 28-37 ° C. The optimum pH for growth is around neutral.

The cells are activated for steroid-1-dehydrogenation by the addition of 1,2-saturated-3-keto steroid, such as androst-4-ene-3,17-dione. cortisone acetate is added in an amount of 0.005% (v / v) or more. In the presence of the inducer, the cells are incubated for 6 hours and then harvested and harvested

I dry them.

The inducer may be added to the microorganisms at any point in the growth cycle. For lard oil (an oil containing triglycerides and free fatty acids)

cultures cultured on similar media begin to synthesize steroid-1-dehydrogenase rapidly, whereas glucose-cultured microorganisms start producing enzymes only after digestion of the substrate. The incubation is continued for 6 hours or more after the inducer is added and the cells are harvested for use in the solvent process or for drying.

The cells are separated from the culture medium for drying and concentrated by conventional methods such as centrifugation, flocculation, filtration or ultrafiltration. The isolated cells are then dried under reduced pressure at 1-85 ° C, preferably 5575 ° C, by spraying or spinning with warm air until the material has a moisture content of 1-10%, preferably 5%. Cells used for bioconversion were stored at 5 ° C. Active dried cells can be prepared by standard methods by immobilizing the dried cells, for example, by encapsulation in a polyacrylamide gel or by collagen or covalent bonding to a polyelectrolyte carrier (Methods in Enzymology, Vol. XLIV, Academic Press Inc., New York, 11- 317 (1976).

Bioconversion is achieved by the interaction of the prepared cells with the steroid substrate. In general, the cells and the steroid are suspended in a weakly buffered aqueous medium of pH 6-10, preferably 7.258.5. Cell concentration ranges from 0.1 to 50 g / l, steroid to cell weight ratio from 0.05 to 5. In aqueous media, a cell concentration of 810 g / l or steroid 5-10 g / l is preferred for bioconversion. A catalytic amount of exogenous electron transfer material (e.g., 5 x 10 ^-4 moles of menadione) is also added to the reaction mixture. The catalysts used include menadione (2-methyl-1,4-naphthoquinone), phenazine methosulfate, dichlorophenylindophenol, 3,4-naphthoquinone, menadione bisulfite, ubiquin45 non (Coenzyme Q) and vitamin K type compounds. The mixtures were incubated for 0-14 days at 5-45 ° C. During the incubation, the mixture must contain an excess of molecular oxygen, so it is advantageous to mix. The rate of 1-dehydrogenation generally decreases with time. Bioconversion using 10 g / L substrate can be 98-100% in less than 24 hours.

The procedure can be performed in the following ways:

a, The steroid and menadione are suspended in a weakly buffered aqueous solution and the dried cells are added. Surfactants similar to Tweewn 8060 can also be added in a concentration of 0-5%,

b) The cells are suspended in an aqueous buffer solution and then electron transfer soluble in methanol, ethanol, acetone (not more than 5% of _the final volume) is added. in dimethylformamide, ethanol, methanol, acetone, dimethyl sulfoxide) or in a water immiscible organic solvent (such as toluene).

c) Rehydrate the dry cells in a small amount of buffer by adding additional buffer or organic solvent. Ethanol, acetone, xylene, butyl acetate, methylene chloride or toluene are added to give a final 0-95% (v / v). The amount of aromatic hydrocarbon used may vary from half of the solvent needed to dissolve the most soluble steroid to four times the solvent needed to dissolve the least soluble steroid. To initiate the reaction, an electron transfer agent and a steroid are added to the reaction mixture. The aromatic carbon hydrogene is preferably added with the steroid, although this may occur at an earlier or later time.

d) The wet cell mass is suspended or the fermentation broth diluted with weakly buffered aqueous solution and an exogenous electron transfer agent, steroid and aromatic hydrocarbon are added.

The compounds useful in the reaction according to the invention belong to the steroids S-keto-α-androstene and 3-keto-α ⁴ -pregnene. The substrates of steroid 1-dehydrogenase are saturated between ring 1 and ring 2 carbon atoms and contain a hydroxyl or keto group at the 3-position of ring A. The androstene type compounds include:

1) androst-4-ene-3,17-dione,

2) androst-4,9 (II) -diene-3,17-dione and these

6-alpha-fluoro, 6-alpha-methyl or 16-methyl,

3) 11-beta-hydroxyandrost-4-ene-3,17-dione

and its derivatives.

Useful compounds include S-keto-α-pregnene compounds. include:

1) 17-alpha-hydroxypregn-4-ene-20-yn-3-one and its 16-methyl derivatives,

2) 11-beta, 21-dihydroxypregn-4,17 (20)-dien-3-one and 6-alpha-methyl derivatives,

3) 20-chloro-pregn-4,9 (11), 17 (20) triene-21-al-3-one,

4) a number of groups of S ?, diketo-a? -Pregnene, i.e.

(a) 11,17,21-trihydroxy compounds such as hydrocortisone and

6-alpha-methyl derivative,

b) 9-beta, 11-beta-epoxy-17,21 compounds such as 9-beta, 11-beta-epoxy-17,21-dihydroxy-15-beta-methyl-pregn-4-ene-3 , 20-dione,

c) 3,20-diketo-4,9 (11) -pergene dienes such as 17-alpha, 21-dihydroxypregn-4,9111) diene-3,20-dione and 16-alpha- methyl, 16-beta-methyl or 16-alpha-hydroxy derivatives or 17-alpha-acetate ester,

d) 3,20-diketo-4,9 (II) -, 4,9 (11), 16-pregnene trienes such as

21-hydroxypregn-4,9 (II), 16-triene-3,20-dione and its 6-alpha-fluoro derivative.

The 2110-ester derivatives of steroids containing the hydroxyl group at positions 21 and 21 can also be used as substrates. Preferred 21-esters contain lower alkyl or aryl groups such as acetic acid or a monocyclic carboxylic acid such as benzoic acid, such as lower fatty acids.

The bioconversion product and the unmodified substrate may be separated from the mixture by conventional means. The steroids are usually isolated by filtration followed by extraction of the cake remaining on the filter with an organic solvent such as acetone or methylene chloride. Alternatively, the whole bioconversion mixture is extracted with a water immiscible organic solvent such as butyl acetate or methylene chloride and the product is separated from the organic solvent.

The following examples illustrate the process of the present invention, but are not intended to limit the invention thereto. The examples also prove that the process of the invention is much better than the methods known in the literature. Bioconversion was performed under the conditions detailed above.

Example 1

Preparation of dried cells

Bacterium cyclooxydans (ATCC 12673) was inoculated into 6-6 g / l starch sugar, peptone and corn jam (pH 7.0) shaking bulls. The cultures were incubated at 28 ° C on a rotary shaker until glucose depletion occurred. At this time, 0.5 g / l of cortisone acetate was added and the flask was incubated for an additional 16 hours. Cells were harvested by centrifugation, washed twice with water, and dried under reduced pressure at 45 ° C.

Bioconversion of 6-alpha-methyl-cortisone

Half grams of the cells prepared above were rehydrated in 50 ml 50 mM phosphate buffer pH 7.5 with mixing. To the cell suspension was added menadione in 0.025 ml / 50 ml in ethanol solution (8.6 mg / l ethanol). The substrate, dissolved in dimethylformamide (DMF) (100 mg 6-alpha-methylhydrocortisone / ml DMF) was added to the final bioconversion mixture at 0.5 g / l. The mixture was incubated at 28 ° C with stirring. After 4 hours of incubation, 91% of the steroids were converted. 6-alpha-methyl-prednisolone was isolated by conventional methods.

Example 2

Preparation of Androsta-1,4-9 (II) -triene-3,17-dione

(a) Production of the biocatalyst

Shake flasks containing 6-6 g / l glucose, corn jam and spray-dried lipid emulsion were cultured with Arthrobacter simplex (ATCC 6946). Cultures were incubated at 28 ° C in a rotary shaker until glucose depletion was observed. Cortisone acetate (0.15 g / L) was then added to initiate steroid-1-dehydrogenase synthesis. After overnight incubation, cells were harvested by centrifugation. Cell pellets were dried at 55 ° C under low pressure for 24 hours. 24 hours later, the dry material was placed in an airtight container and stored at 5 ° C until used for bioconversion.

b) Bioconversion of androst-4,9 (II) -diene-3,17-dione in g / l of dried cell is 50 mM,

Phosphate pH 7.5 was rehydrated with stirring for 30 minutes. Thereafter, the amount of menadione in the form of a dry powder was added to the cell suspension as needed to reach a final concentration of 86 mg / L. The micronized androst-4,9 (II) -diene-3,17-dione was suspended in dimethylformamide and added to the reaction mixture to give 2.5 g of steroid / l and 2% (v / v) dimethylformamide. The mixture was incubated at 31 ° C with stirring in the presence of air for 24 hours. After completion of the incubation, androst-1,4,9 (II) -triene-3,17-dione was isolated by conventional methods.

Example 3

The following compounds were converted to their 1,2-dehydro derivatives using A. simplex:

1st androsta-4-on-3,17-prize

2. 6-alpha-fluoro-androst-4,9 (11) - di-3,17-di

3. 6-alpha-methyl-androst-4,9 (11) -diene-3,17-dione

4. 16-beta-methyl-androst-4,9 (11) -diene-3,17-dione

5. 17-alpha-hydroxy-pregn-4-ene-20-yn-3-one

6. 17-alpha-hydroxy-pregn-4,9 (11) -dien-20-yn-3-one

7. 17-alpha-hydroxy-16-beta-methyl-pregn-4,9- (11,11-diene-20-yn-3-one; 8,11-beta, 21-dihydroxy-pregn-4,17 ( 20) -diene-3-one

9. 21-Acetoxy-11-beta-hydroxy-pergn-4,17- (20)-dien-3-one

10. 6-alpha-methyl-11-beta, 21-dihydroxy-pregn-3,17 (20)-dien-3-one

11. 20-Chloro-pren-3,9 (11,17 (20) -triene-21-al-3-one)

12. hydrocortisone

13. 6-alpha-methylhydrocortisone

14. 21-Acetoxy-11-beta, 17-dihydroxy-16-beta-methyl-pregn-4-ene-3,20-dione

15. 21-Acetoxy-9-alpha-fluoro-11-beta, 17-dihydroxy-16-beta-methyl-pregn-4-ene-3,20-dione

16. 21-Acetoxy-9-beta, 11-beta-epoxy-17-hydroxy-16-beta-methyl-pregn-4-ene-3,20-dione

17. 21-Acetoxy-17-hydroxy-pregn-4,9 (11)-diene-3,20-dione

18. 21-Acetoxy-16-alpha, 17-dihydroxy-pregn-4,9 (II)-diene-3,20-dione

19. 21-Acetoxy-17-hydroxy-16-alpha-methyl-pregn-4,9 (11)-diene-3,20-dione

20. 21-Benzyloxy-17-hydroxy-16-beta-methyl-pregn-4,9 (II)-diene-3,20-dione

21. 21-Acetoxy-17-hydroxy-16-beta-methyl-pregn-4,9 (II)-diene-3,20-dione

22. 21-Acetoxy-pregn-4,9 (11), 16 (17) -triene-3,20-dione

23. 21-Acetoxy-6-alpha-fluoro-pregn-4,9 (11), 16-triene-3,20-dione.

The conversion resulted in the following products:

androsta-1,4-diene-3,17-dione

2a 6-alpha-fluoro-androsta-1,4,9 (II) -triene-3,17-dione

3α 6-alpha-methyl-androsta-1,4,9 (II) -triene-3,17-dione

4a 16-Beta-Methyl Androsta-1,4,9 (II) -triene-3,17-dione

5α-17-alpha-hydroxy-pregn-1,4-diene-20-yn-3-one

6α 17-alpha-hydroxypregn-1,4-9 (11L) -trien-20-yn-3-one

7a 17-alpha-hydroxy-16-beta-methyl-pregn-1,4-9 (11) -trien-20-yn-3-one

8α, 11β-beta, 21-dihydroxypregn-1,4,17 (20, -trien-3-one

9a-21-acetoxy-11-beta-hydroxypregn-1,4,17- (20) -trien-3-one and 11-beta, 21-dihydroxypregn-1,4,17 (20) -triene- 3-one

10α-6-alpha-methyl-11-beta, 21-dihydroxy-pregn-1,4,17 (20) -trien-3-one

11a 20-Chloro-pregn-1,4,9 (11), 17 (20) -tetraen-21-al-3-one

12a prednisolone

13a 6-alpha-methyl-prednisolone

14a 21-acetoxy-11-beta, 17-dihydroxy-16-beta-methyl-pregn-1,4-diene-3,20-dione and 11-beta, 17,21-trihydroxy-16-beta-methyl-pregn. l, 4-diene-3,20-dione

-510

15a 21-acetoxy-9-alpha-fluoro-11-beta, 17-dihydroxy-16-beta-methyl-pregn-1,4-diene-3,20-dione and 9-alpha-fluoro-11-beta, , 21-trihydroxy-16-beta-methyl-pregn-l, 4-diene-3,20-dione

16α 21-Acetoxy-9-beta, 11-beta-epoxy-17-hydroxy-16-beta-methyl-pregn-1,4-diene-3,20-dione and 9-beta, 11-beta-epoxy-17 , 21-dihydroxy-16-beta-methyl-pregn-l, 4-diene-3,20-dione

17α-21-acetoxy-17-hydroxy-pregn-1,4,9 (11) -

-triene-3,20-dione and 17,21-dihydroxypregn-1,4,9 (11) -triene-3,20-dione

18a 21-acetoxy-16 alpha, 17-dihydroxypregn-1,4,9 (11) -triene-3,20-dione and 16 alpha, 17,21-trihydroxypergn-1,4,9 (ll) -triene-3,20-dione

19a 21-Acetoxy-7-hydroxy-16-alpha-methyl-pregn-1,4,9 (11) -triene-3,20-dione and 17,21-dihydroxy-16-alpha-methyl-pr eg η-1,4,9 (11) - trene-3,20-dione

20a 21-Benzyloxy-17-hydroxy-16-beta-methyl-pr e g η-1,4,9 (11) -trin-3,20-dioxo

21a 21-acetoxy-17-hydroxy-16-beta-methyl-pregn-1,4,9 (11) -triene-3,20-dione and

17,21-dihydroxy-16-beta-methyl-pregn-l, 4,9 (ll) -triene-3,20-dione

22a 21-acetoxypregn-1,4,9 (11), 16-tetraene 5 -3,20-dione and 21-hydroxypregn-1,4,9- (11), 16-tetraene 3,20- dion

23α-21-acetoxy-6-alpha-fluoro-pregn-1,4,9 (11), 16-tetraene-3,20-di and 6-alpha-fluoro-21-hydroxy-pregn-1,4,9 (11), 16-tet10 Rene-3,20-dione.

Example 4

Two different steroids were transformed with A. simplex and dried A. simplex cells in the fermentation broth. After each bioconversion was considered complete (indicated by the stop of residual substrate level reduction), the steroid in the mixture was extracted and isolated to give the typical conditions of both processes. The following table summarizes these results.

Comparison of yields of different types of bioconversions

Szubsztrá- tum Kind of bioconversion Useful steroid recovery (56) (a) % Δ * compound % remaining remarks ing Of formula (I) fermentation 77.2 1st Generation 2.6 87.4 weak 1-de- compound 4- 2.8 2nd Generation 10.4 83.6 hydrogenation 10 g / l dried cell 82.6 1st Generation + 9.9 2nd Generation · 100.0 95.0 5.0 Compound of Formula II fermentation 18.1 1st Generation Generation 2 was not received 59.1 40.9 (B) Et g / l dried cell 79.6 1st Generation 2nd generation we did not get 97.8 2.1

(a) = The useful steroid is either used as a Δ * compound in further syntheses or is fed back to another bioconversion as a 1,2-dihydro substrate to produce the particular product.

(b) = Significant amounts of other undesirable steroid molecules were also recovered.

Example 5

Arthrobacter simplex was cultured in a 5-liter .MicrofernT fermenter, induced for steroid-1-dehydrogenase synthesis, and harvested by centrifugation. The details of the cell paste obtained were used in two different methods. In the first procedure, the cells were pressurized under reduced pressure at 55 ° C. The second procedure utilized the method proposed in U.S. Patent No. 3,360,439,665, wherein the cells were dried with acetone. The cells were mixed with acetone and dried under reduced pressure at 5 ° C. The dried sejtkészitményeket was then used for the bioconversion of ^9ill -androstane-dione, so that the conversion of the mixture of cell concentration was 10 g / 1, the steroid concentration of 10 g / 1st

-612

Comparison of bioconversion activity of A. simplex cells dried by various methods

cell Type Time of measurement (hours) % remaining substrate Bioconversion Activity (+) Product formed g

hours / g cell

acetone 1 98.7 0013 dried 4 95.5 0011 24 84.1 * 0007 Heat count 1 85.1 0149 powdered 4 23.2 0192 24 7.2 * 0039

(+) = Bioconversion activity was calculated as follows:

% product formed x starting substrate

= g of product formed

100 products formed (g) / incubation time (h) / cell added (g) = activity (*) = Residual levels are not reduced by further incubation.

Example 6

The 6-alpha-methylhydrocortisone of Example 1 or the androst-4,9 (II)-diene-3,17-dione of Example 2 were substituted with the corresponding substituents to give the following products.

substrates:

1. 21-Acetoxy-9-alpha-fluoro-11-beta, 16-alpha, 17-trihydroxy-pregn-4-ene-3,20-dione

2. 21-Acetoxy-6-alpha, 9-alpha. difluoro-11-beta, 16-alpha, 17-trihydroxy-pregn-4-ene-3,20-dione-16,17-acetonide

3. 21-Acetoxy-6-alpha-fluoro-11-beta-hydroxy-16-alpha-methyl-pregn-4-ene-3,20-dione

4. 21-Acetoxy-6-alpha-fluoro-11-beta, 17-hydroxy-pregn-4-ene-3,20-dione

5. 21-Acetoxy-6-alpha, 9-alpha-difluoro-11-beta, 17-dihydroxy-16-methyl-pregn-4-ene-3,20-dione

6. 21-Acetoxy-9-alpha-fluoro-11beta, 16-alpha, 17-trihydroxypregn-4-ene-3,20-dione-16,17-acetonide

7. 21-Acetoxy-9-beta, 11-beta-epoxy-6-alpha-fluoro-16-alpha, 17-dihydroxy-pregn-4-ene-3,20-dione-16,17-acetonide

8. 21-Acetoxy-9-beta, 11-beta-epoxy-16-alpha-hydroxy-pregn-4-ene-3,20-dione

9. 21-Acetoxy-9-beta, 11-beta-epoxy-16-alpha, 17-dihydroxy-pregn-4-ene-3,20-dione-16,17-acetonide.

Products:

1. 21-Acetoxy-9-alpha-fluoro-11-beta, 16-alpha, 17-trihydroxy-pregn-1,4-diene-3,20-dione and 9-alpha-fluoro-11-beta, 16- alpha, 17,21-tetrahydroxy-ene-l, 4-diene-3,20-dione

2. 21-Acetoxy-6-alpha, 9-alpha-difluoro-11-beta-4α, 16-alpha, 17-trihydroxypregn-1,4-diene-3,20-dione-16,17- acetonide and 6-alpha, 9-alpha-fluoro-11beta, 16-alpha, 17,21-tetrahydroxypregn-1,4-diene-3,20-dione-16,17-acetonide

3. 21-Acetoxy-6-alpha-fluoro-11-beta-hydroxy-16-alpha-methyl-pregn-1,4-diene-3,20-dione and 6-alpha-fluoro-11-beta, 21- dihydroxy-16-alpha-methyl-pregn-l, 4-diene-3,20-dione

4. 21-Acetoxy-6-alpha-fluoro-11-beta, 17-di50-hydroxy-pregn-1,4-diene-3,20-dione and 6-alpha-fluoro-11-beta, 17,21-trihydroxy. l, 4-diene-3,20-dione

5. 21-Acetoxy-6-alpha, 9-alpha-difluoro-11-beta, 17-dihydroxy-16-alpha-methyl-pregn-1,455-diene-3,20-dione and 6-alpha, 9- alpha-difluoro-ll-beta-17,21-trihydroxy-16-alpha-methyl-l, 4-diene-3,20-dione

6. 21-Acetoxy-9-alpha-fluoro-11-beta, 16-alpha, 17-trihydroxy-pregn-1,4-diene-3,20-di60 on-16,17-acetonide

7. 21-Acetoxy-9-beta, 11-beta-epoxy-6-alpha-fluoro-16-alpha, 17-dihydroxy-pregn-1,4-diene-3,20-dione-11,17-acetonide

8. 21-acetoxy-9-beta, 11-beta-epoxy-16-alpha65-hydroxypregn 1,4,4-diene-3,20-dione and 98

-beta, beta-ll-epoxy-16-alpha, 21-dihydroxy-pregn-l, 4-diene-3,20-dione

9. 21-Acetoxy-9-beta, 11-beta-epoxy-16-alpha, 17-dihydroxy-pregn-1,4-diene-3,20-dione-16,17-acetonide. 5

Example 7 Bioconversion of 11-beta-Hydroxy-androst-4-ene-3,17-dione

One gram of dried A. simplex cell prepared as described in Example 2 was suspended in 100 ml of 50 mM phosphate buffer, pH 7.5, 15, and menadione dissolved in 3A ethanol (2-3% methanol denatured in ethanol) was added. mg / l to a final concentration. Half a gram of 11-beta-hydroxyandrostenedione was then added and incubated in a rotary mixer at 31 ° C. After one day of incubation, two products were observed by thin layer chromatography on methylene chloride extract. The steroid should take approx. 95% was present in the form of 11-25-beta-hydroxy-an d-rostal, 4-diene-3,17-dione. The remainder was an unmodified substrate.

Example 8 ·

Following the procedure of Example 7, starting from the following androstenedione derivatives, the following 1-dehydrogenated product 35 was obtained:

Substrate -> Product

H-beta-hydroxy-16-beta-ethyl-androst-40

-4-ene-3,17-dione-> 11-beta-hydroxy-16-beta-methyl-androst-1,4-diene-3,17-dione

2. 11-beta-hydroxy-16-alpha-methyl-androst-4-ene-3,17-dione -> 45,11-beta-hydroxy-16-alpha-methyl-androst-1,4-diene-3 , 17-dione;

3. 6-alpha-fluoro-11-beta-hydroxy-androst-4-ene-3,17-dione-> 6-alpha-fluoro-11-beta-hydroxy-androst-50

l, 4-diene-3,17-dione

4. 6-alpha-methyl-11-beta-hydroxyan drosta-4-ene-3,17-dione->

6-alpha-methyl-11-beta-hydroxyandrosta-1,4-diene-3,17-dione ·

5. 11-alpha-hydroxyandrost-4-ene-3,17-dione-

ll-alpha-hydroxy-androsta-l, 4-diene-3,17-dione

6. androst-4-ene-3,11,17-trione-

androsta-l, 4-diene-3, ll, 17-trione.

The use of 1,2-dihydro steroids is well known. For example, U.S. Pat. No. 3,284,447 discloses that a * .4.9Ui) -pregnene trienes can be used to synthesize diuretic corticosteroids substituted on carbon 16. U.S. Pat. No. ^4,041,055 describes the synthesis of corticosteroids from the ^{Δ 4} -androstenedione, thereby demonstrating that 1,2-dihydroandrostenes are important intermediates in the preparation of pharmaceutically useful steroids.

Claims (5)

PATENT CLAIMS A process for converting 1,2-saturated-3-keto steroids with pregnane or androstane backbones to 1,2-dehydro steroids, characterized in that the 1,2-saturated steroids are heat-dried at least 40 ° C in Arthrobacter or Bacillus genus 1-dehydrogenating microorganism. The method of claim 1, wherein the heat-dried microbial cells contain from 1% to 10% moisture. The process according to claim 1, wherein the 1-dehydrogenating microorganism is Arthrobacter simplext or Bacteriura cyclooxydans. The process of claim 1, wherein the 1,2-saturated steroids are transformed with bacterial cells of heat-dried 1-dehydrogenating microorganisms in the presence of an exogenous electron transfer agent. The process according to claim 4, wherein the exogenous electron transfer agent is 2-methyl-1,4-naphthoquinone, phenazine methosulfate, dichlorophenolindophenol, 1,4-naphthoquinone, 2-methyl-1,4 naphthoquinone bisulfite, ubiquinones (Coenzyme Q) or vitamin K type compounds.